1. Field of the Invention
The present invention relates to the field of molecular biology and nucleic acid chemistry. More specifically, it relates to methods and reagents for detecting and distinguishing herpes simplex virus (HSV) types 1 and 2, Treponema pallidum, and Haemophilus ducreyi.
2. Description of Related Art
HSV, T. pallidum, which is the causative agent of syphilis, and H. ducreyi, which is the causative agent of chancroid, are the three primary causative agents of genital ulcerative diseases in the United States. Currently, the diagnosis of genital ulcerative disease is based predominantly on the clinical presentation of the ulcer itself. However, the diagnosis of genital herpes, syphilis, or chancroid is made difficult by the overlapping patterns of clinical presentation and the occurrence of multiple or mixed infections (see Sturm et al., 1987, Microbiol. Rev. 63:98-101). Because specific and distinct therapies are required for the treatment of infections with each of these agents, diagnostic tests are needed which accurately identify and distinguish the causative agents of genital ulcers.
HSV normally is detected by viral culture. However, the sensitivity of detection depends on the stage and duration of the lesion. Although the sensitivity of detection by viral culture from vesicular lesions approaches 100%, the sensitivity of detection from crusted or healing lesions drops to 30%.
T. pallidum normally is detected by one of two methods. The standard immediate test is by darkfield microscopic examination of lesion material. Alternatively, T. pallidum infection is identified by the presence of specific antibodies. Darkfield examination has several disadvantages. Darkfield examination requires specialized equipment and an experienced technician, and only provides a detection sensitivity of less than 75%. Similarly, serological detection is of limited usefulness. Detectable antibodies may not appear for a week after the appearance of a lesion and can persist from a previous infection. Furthermore, the assay may require multiple patient visits.
H. ducreyi normally is detected by culture. The assay, which is not widely available, only provides a sensitivity of less than 70%.
The invention of the polymerase chain reaction (PCR), a method for amplifying specific sequences of nucleic acids, makes possible the rapid detection of specific nucleic acid sequences present in a sample in what was previously an undetectably low quantity (see U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188, each of which is incorporated herein by reference). Direct detection of an amplified nucleic acid sequence by hybridization with a sequence-specific oligonucleotide probe makes possible the detection of etiologic agents contained in a sample, thereby enabling rapid and sensitive diagnostic assays.
Current PCR-based assays for HSV, T. pallidum, and H. ducreyi are designed to detect only a single target. A rapid and sensitive diagnostic test assay capable of detecting and identifying HSV types 1 and 2, T. pallidum, and H. ducreyi in possibly multiply-infected samples has not be described.